Fiber-Optic Chemical Sensorxe2x80x9d Anal. Chem. 68:1748 (1996). This sensor was constructed by holding a small amount of an internal reagent solution at the tip of a fiber-optic bundle with a piece of gas-permeable membrane. Nitric oxide diffuses across the membrane into this internal solution, where a chemiluminescent reaction between nitric oxide, hydrogen peroxide, and luminol takes place. The drawbacks of this sensor include the following: 1) the response time (approximately 8-17 seconds) is longer than the time needed for nitric oxide in the solution to be converted to nitrite; 2) the detection of nitric oxide is complicated by interferences from dopamine, uric acid, ascorbic acid, and cysteine, 3) the sensor is relatively large in size (greater than 6 mm in diameter) and thus difficult to use for the measurement of cellular nitric oxide levels (and impossible for intracellular measurements); and 4) the sensor has relatively poor sensitivity, i.e., a relatively high limit of detection (approximately 1.3 mM of nitric oxide).
Sensors involving sol-gel technology have also been attempted. The process involves hydrolyzing an alkoxide of silicon to produce a sol, which then undergoes polycondensation to form a gel. Biomolecules are immobilized by being entrapped in the sol-gel. In one case, horse-heart cytochrome c was encapsulated in a sol-gel and absorbance-based spectral shifts were used to monitor the binding of nitric oxide. See Blyth et al., xe2x80x9cSol-Gel Encapsulation of Metalloproteins for the Development of Optical Biosensors for Nitrogen Monoxide and Carbon Monoxidexe2x80x9d Analyst 120:2725 (1995). Unfortunately, the sensor reaction is reported to have taken two hours to reverse, making dynamic measurements impossible.
What is needed is a sensor of relatively small size and good sensitivity that measures nitric oxide with little or no interference from other analytes in a short enough time period to permit dynamic measurements.
The invention relates generally to optical sensors, methods of sensor fabrication and uses of such sensors, and more particularly the use of such sensors for the detection if nitric oxide. The present invention contemplates both fiber-optic sensors and optical fiberless sensors comprising nitric oxide-binding compounds, such compounds permitting the specific binding of nitric oxide (e.g., non-covalent binding) with little or no interference from other analytes.
With regard to fiber-optic sensors, the present invention contemplates an optical fiber having a fiber tip, said tip comprising an immobilized nitric oxide-binding compound. It is not intended that the present invention be limited by the means by which the nitric oxide-binding compound is immobilized. In one embodiment, the tip of the fiber is treated so as to have reactive groups and the nitric oxide-binding compound is covalently linked directly to the fiber via the reactive groups. In another embodiment, the tip has an inert coating (i.e., inert relative to nitric oxide) such as a metal layer (preferably, a non-linear layer and more preferably, spheres comprising metal) and the nitric oxide-binding compound is immobilized on the metal layer. In a preferred embodiment, the tip is treated to create reactive groups (e.g., thiol groups), spheres of metal colloid are attached to the tip via the reactive groups, and the nitric oxide-binding compound is immobilized on the metal colloid spheres.
It is not intended that the present invention be limited to the nature or dimensions of the metal layer. A variety of metals and metal colloids are contemplated, including but not limited to, colloids of gold, silver, tungsten, thoriasol, antimony pentoxide, carbon, red iron oxide, titanium dioxide and platinum (available commercially from Vector Laboratories, Inc., Burlingame, Calif.; Nanoprobes, Inc., Stony Brook, N.Y.; and Polysciences, Inc., Warrington, Pa.). In a preferred embodiment, the metal layer is a monolayer of spheres comprising gold colloid, said spheres attached to an end of a fiber as a substrate for spontaneous attachment of the nitric-oxide-binding compound. While not limited to particular dimensions, the size of the gold colloid does produce a marked difference in the fluorescence intensity measured. The present invention contemplates colloid sizes (and in particular gold colloid sizes) ranging from very small, 2 nm, to very large, 250 nm (and more preferably, between 5 nm and 100 nm), said colloids immobilized on the end of a fiber to provide a base for protein attachment. While a precise understanding of the mechanism for this phenomenon is not necessary in order to practice the invention, it is surmised the intensity changes seen in the fluorescence emission are not a result of surface coverage, and availability of sites for protein adsorption, but instead a quenching or enhancement by the gold itself. In general, the optimum fluorescence is achieved with particles sizes of approximately 100 nm.
In another embodiment, the nitric oxide-binding compound is a porphyrin group- or heme group-containing protein. In another embodiment, the nitric oxide-binding compound is a heme-binding protein. Regardless of whether the protein is a heme-group-containing protein or a heme-binding protein, in one embodiment, the present invention contemplates that the protein (or peptide) is dye-labeled (e.g., with dyes which can be used for protein labeling that do not react to nitric oxide, such as Oregon Green dyes). This has been found to increase the signal to noise ratio of the sensors of the present invention.
It is not intended that the present invention be limited to specific heme-group-containing proteins. The heme-group-containing proteins are limited only in the respect that they bind nitric oxide, and more preferably, they bind nitric oxide specifically (i.e., they do not bind interfering substances). The preferred heme-group-containing protein is cytochrome cxe2x80x2 (as distinct from cytochrome c). It is not intended that the present invention be limited to the source of cytochrome cxe2x80x2. Nonetheless, preferred sources include, but are not limited to, microorganisms, more preferably bacterial sources, and more particularly, purple phototropic bacteria, aerobic nitrogen-fixing bacteria, and facultatively denitrifying bacteria, and still more particularly, sources such as C. vinosum, R. purpureus, and R. gelatinosa. 
Insects have been shown to have both heme group-containing proteins that bind nitric oxide (M. C. Ribeiro et al., xe2x80x9cReversible Binding of Nitric Oxide by a Salivary Heme Protein from a Bloodsucking Insect,xe2x80x9d Science 260:539 (1993); J. G. Valenzuela et al., xe2x80x9cA Salivary Nitrophorin (Nitric-Oxide-Carrying Hemoprotein) In The Bedbug Cimex lectularius,xe2x80x9d J. Exper. Biol. 198:1519 (1995)], as well as heme-binding proteins [P. L. Oliveira et al., xe2x80x9cA Heme-binding Protein from Hemolymph and Oocytes of the Blood-sucking Insect, Rhodnius prolixus,xe2x80x9d J. Biol. Chem. 270:10897 (1995)]. The present invention contemplates both groups of proteins as useful in the preparation of optical sensors.
It is not intended that the present invention be limited to specific heme-binding proteins. The heme-binding proteins are limited only in the respect that they bind nitric oxide, and more preferably, they bind nitric oxide specifically (i.e., they do not bind interfering substances). The preferred heme-binding protein is the heme-binding protein isolated and characterized from both the hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. 
The present invention also contemplates sensors without binding compounds. More specifically, the present invention contemplates a sensor based on analyte adsorption to a metal surface reported by fluorescence changes of an attached dye molecule. It is also not intended that the present invention be limited by the nature of the particular dye. In one embodiment, said dye is a fluorescein or fluorescein derivative adsorbed to a metal (e.g. gold) surface. In another embodiment, diaminofluorescein is adsorbed to a gold surface. In a preferred embodiment, difluorofluorescein is adsorbed onto a gold surface.
The invention also contemplates optical fiberless sensors capable of detecting nitric oxide. The sensors of the present invention are: (1) small enough to enter a single mammalian cell relatively non-invasively, (2) fast and sensitive enough to catch even minor alterations in the concentration of nitric oxide and (3) mechanically stable enough to withstand the manipulation of the sensor to specific locations within the cell. Importantly, the fiberless sensors of the present invention are non-toxic and permit the simultaneous monitoring of several cellular processes.
In one embodiment, the present invention contemplates fiberless optical sensors comprising a nitric oxide-binding compound. It is not intended that the present invention be limited by the precise composition of the fiberless sensors. The fiberless sensors of the present invention are either solid or semisolid particles ranging in size between approximately 1 micrometer and 1 nanometer in diameter, and more preferably, between 5 nanometers and 250 nanometers. The ultimate small size is attained by fine grinding and filtering or by micro-emulsion techniques used to form mono-disperse colloidal particles (rather than nano-fabrication). In one embodiment, the sensor is selected from the group consisting of polymer fiberless sensors, acrylamide fiberless sensors, sol-gel fiberless sensors and metal fiberless sensors.
In one embodiment, the polymer fiberless sensors of the present invention comprise a nitric oxide-binding compound (such as a porphyrin) and a polymer. It is not intended that the present invention be limited to a particular polymer. In one embodiment, the polymer is selected from the group consisting of poly(vinyl chloride), poly(vinyl chloride) carboxylated and poly(vinyl chloride-co-vinyl acetate-co-vinyl alcohol). In a particular embodiment, the polymer fiberless sensors further comprise an additive and a plasticizer.
In one embodiment, the acrylamide fiberless sensors of the present invention comprise polyacrylamide and a nitric oxide-binding compound. In a preferred embodiment, the acrylamide fiberless sensors further comprise N,N-methylenebi-(acrylamide) and the mixture is polymerized to a gel.
In one embodiment, the sol-gel fiberless sensors of the present invention comprise a nitric oxide-binding compound entrapped in a matrix, such as a silica sol. Where the compound is a protein, stabilizers can be used. The gels are typically aged before use.
In one embodiment, the metal fiberless sensors of the present invention comprise a nitric oxide-binding protein (or peptide) in combination with a metal selected from the group consisting of gold, silver, platinum and alloys thereof (e.g., a gold/silver alloy). In one embodiment, the protein (or peptide) is dye-labeled (e.g, with Oregon green 514). Typically, such metal fiberless sensors are made by combining between 0.01% and 1% , and more preferably approximately 0.1% protein or peptide (by weight) in colloid solution. Spontaneous adsorption of the protein to the metal surface takes place within minutes.
The present invention also contemplates fiberless sensors with attached dyes, including but not limited to metal fiberless sensors. In one embodiment, the metal fiberless sensors of the present invention comprise a fluorescein derivative dye attached to colloidal gold. It is not intended that the present invention be limited by the type of fluorescein derivative dye. In one embodiment, said fluorescein derivative dye is 4-carboxy-2xe2x80x2,7xe2x80x2,-difluorofluorescein, succinimidyl ester. It is not intended that the present invention be limited by the geometry of the colloidal gold applied to the sensor tip. In one embodiment, said colloidal gold is 50 nm in thickness. It is not intended that the present invention be limited by the geometry or preparation of the metal fiberless sensor. In one example, said metal fiberless sensor are 100 xcexcm core diameter multimedia fibers. In another example said sensors are prepared from 0.5xc3x974.5 cm2 quartz slides.
In one embodiment, said metal fiberless sensors of the present invention comprising a fluorescein derivative dye attached to colloidal gold are coupled with reference microspheres. It is not intended that the present invention be limited to the type of reference microsphere. In one embodiment, 40 nm fluorescent carboxylate-modified polystyrene microspheres with 488 nm excitation and 685 nm emission are coupled to the sensor tip.
It is not intended that the present invention be limited by the manner in which the sensors of the present invention are introduced into cells. In one embodiment, a buffered suspension of fiberless sensors is injected into the sample cell with a commercially-available pico-injector. In another embodiment, the fiberless sensors of the present invention are shot into a cell with a commercially-available particle delivery system or xe2x80x9cgene gunxe2x80x9d (such gene guns were developed and are now routinely used for inserting DNA into cells). In other embodiments, the fiberless sensors of the present invention are remotely steered into a cell, by photon pressure or xe2x80x9claser tweezersxe2x80x9d. This uses an infra-red laser beam which traps the particles or magnetically, by remotely steering magnetic nanoparticle pebbles (commercially available) into a cell.
It is also not intended that the present invention be limited by the detecting means. In one embodiment, the fiberless sensors of the present invention are addressed by laser beams (rather than fibers), and their fluorescent signals are collected and analyzed by procedures identical to those used for the fiber-tip nanosensors. See U.S. Pat. Nos. 5,361,314 and 5,627,922 to Kopelman et al., hereby incorporated by reference.